NO donor compounds, their preparation, pharmaceutical compositions and uses

Abstract

The present invention discloses a NO donor compound and its preparation method, pharmaceutical composition and use. The structure of this compound is as shown in Formula I. This NO donor compound and its pharmaceutical composition can be prepared as a drug for treating myocardial hypoxic injury disease as a hypoxia-activated NO donor, and the prepared drug can exert its medicinal effect at both cell and animal levels, and the synthesis of this compound is simple and easy to operate.

Description

[Technical field]

[0001] The present invention relates to an NO donor compound, its preparation method, pharmaceutical composition and use, and in particular to an NO donor compound which can be produced as a drug for treating myocardial hypoxic injury diseases, and its preparation method, pharmaceutical composition and use. [Background technology]

[0002] Coronary heart disease is myocardial damage caused by myocardial tissue ischemia (hypoxia at the cellular level) due to organic stenosis or blockage of the coronary artery, and is also called ischemic heart disease. According to a data report released by the China Cardiovascular Center in 2020, the number of people suffering from cardiovascular diseases such as coronary heart disease in China has already reached 290 million. Currently, the high incidence and mortality rate of coronary heart disease poses a serious threat to the health of the people. Nitric oxide (NO) donor drugs are often used to treat coronary heart disease caused by ischemia because they release exogenous NO molecules in the body to promote vasodilation. However, traditional drugs lack selectivity for hypoxia and cannot concentrate in hypoxic areas to release sufficient amounts of NO, resulting in serious side effects.

[0003] In recent years, hypoxia-activated prodrugs have attracted widespread attention, which can reduce off-target toxicity by promoting the selective release of active drug groups in hypoxic microenvironments. Considering that hypoxia is an important factor in causing myocardial injury, using this design concept in the structural design of NO donor drugs to obtain hypoxia-activated prodrugs is an effective way to improve the therapeutic efficacy of such drugs and reduce side effects. Nitroreductase (NTR) is present in mammalian heart, liver, kidney, lung, and brain tissues, which can catalyze the reduction of nitroaromatic compounds by providing hydrogen from NADPH and NADH under anaerobic conditions. Therefore, nitroaromatic compounds are recognized hypoxia-activated groups and may be used in the design of hypoxia-activated prodrugs. This is because NTR in hypoxic cells selectively reduces the nitro group on the aromatic ring and further promotes the release of the active drug group from the prodrug by intramolecular electron transfer. NTR is highly expressed in hypoxic cells and can effectively promote the intensive release of active drug groups in prodrugs in hypoxic microenvironments. Therefore, taking advantage of the hypoxic activation property of nitroaromatic compounds, they can be combined with NO donors to obtain prodrugs that can release sufficient amounts of NO under hypoxic activation, which is of great significance in the treatment of cardiovascular diseases such as coronary heart disease. Summary of the Invention [Problem to be solved by the invention]

[0004] Objective of the invention: In order to solve the problems of poor therapeutic effect and significant side effects of conventional NO donor drugs in treating myocardial hypoxic injury, the present invention aims to provide an NO donor compound that can effectively improve myocardial hypoxic injury in a hypoxic microenvironment, as well as its preparation method, pharmaceutical composition and use. [Means for solving the problem]

[0005] Technical solution: In the first aspect of the present invention, the NO donor compound of the present invention has the structure of formula I: JPEG2025506061000002.jpg55135, where R 1 is a nitro group, R 2 is hydrogen, halogen or a C1-C4 alkyl group; R 3 is hydrogen, halogen or a C1-C4 alkyl group, R 2 is a nitro group, R 1 is hydrogen, halogen or a C1-C4 alkyl group; R 3 is hydrogen, halogen or a C1 to C4 alkyl group.

[0006] The present invention provides a hypoxia-activated NO donor compound by attaching a hypoxia-activated nitroaromatic compound to an NO donor molecule via a linking group.

[0007] Preferably, in the structure: R 1 is a nitro group, and R 2 is hydrogen, halogen or a methyl group; R 3 is hydrogen.

[0008] More specifically, the NO donor compound is JPEG2025506061000003.jpg109170.

[0009] In the second aspect of the present invention, the method for producing the above-mentioned NO donor compound is to obtain compound I by acylation of compound 1 with compound 2, and the specific synthesis steps are as follows:

[0010] (1) 4-hydroxy(N-methyl)aniline hemisulfate is reacted with NaNO2 in the presence of acetic acid to obtain compound 1, and in step (i), the reaction temperature is 0°C, the solvent is acetic acid (AcOH), and the reaction time is 3 h; (2) A benzyl alcohol derivative and bis(trichloromethyl)carbonate (triphosgene, BTC) are reacted in the presence of N,N-diisopropylethylamine (DIPEA) to obtain compound 2, and in step (ii), the reaction temperature is room temperature, the solvent is tetrahydrofuran (THF), and the reaction time is 5 h; (3) Compound 1 and compound 2 are reacted in the presence of N,N-diisopropylethylamine to obtain product I, and in step (iii), the reaction temperature is room temperature, the solvent is dichloromethane (DCM), and the reaction time is 5 h.

[0011] JPEG2025506061000004.jpg51170, where R 1 , R 2 , R 3 The definition is as stated above.

[0012] In a third aspect of the present invention, the pharmaceutical composition of the present invention comprises the NO donor compound and a pharma- ceutically acceptable carrier.

[0013] The NO donor compound can be added to a pharma- ceutically acceptable carrier to prepare a common pharmaceutical preparation, such as a tablet, capsule, syrup, suspension or injection, and the preparation can contain commonly used pharmaceutical auxiliaries such as flavorings, sweeteners, liquid / solid fillers, diluents, etc.

[0014] In the fourth aspect of the present invention, the NO donor compound and pharmaceutical composition of the present invention can be manufactured as a drug for treating myocardial hypoxic injury-related diseases, and the compound is a hypoxia-activated NO donor compound and is used to improve the survival rate of myocardial cells under hypoxic conditions, specifically for treating coronary heart disease. The compound can release NO into cells in a hypoxic microenvironment, and effectively improve myocardial hypoxic injury. Effect of the Invention

[0015] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages:

[0016] (1) Such NO donor compounds and pharmaceutical compositions can effectively release NO under hypoxic conditions, with the amount of NO released reaching up to 40% or more, and can improve the survival rate of cardiomyocytes. They can also function as hypoxia-activated NO donors both in vivo and in vivo. (2) Such NO donor compounds and pharmaceutical compositions are widely used and can be manufactured as drugs for treating myocardial hypoxic injury diseases, and the drugs can exert their medicinal effects at both the cellular and animal levels and have superior therapeutic effects; (3) The compound can be produced by a simple method and is easy to operate. [Brief description of the drawings]

[0017] [Figure 1a] This shows the improving effect of the compound on the survival rate of cardiomyocytes H9c2 under normoxic conditions. [Figure 1b] This shows the improving effect of the compound on the survival rate of cardiomyocytes H9c2 under hypoxic conditions. [Diagram 2] 1 shows the NO release level in hypoxic model mouse hearts for compound I1. [Diagram 3] Expression levels of mTORC1 and TSC2-P proteins in mouse hearts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The technical solution of the present invention will be further described below in conjunction with the examples.

[0019] Example 1: Preparation of N-methyl-N-nitroso-p-phenol (Compound 1) JPEG2025506061000005.jpg47130

[0020] Under light-proof conditions, 4-hydroxy(N-methyl)aniline hemisulfate (1.722 g, 10.0 mmol) was added to 50 mL of acetic acid and dissolved by ultrasonication. A solution of sodium nitrite (1.380 g, 20.0 mmol) dissolved in 10 mL of water was cooled in an ice-water bath and added dropwise to the acetic acid solution. After the addition was completed, the reaction was continued for 3 h in an ice-water bath. After the reaction was completed, the reaction solution was transferred to a beaker, 50 mL of water was added, the pH was adjusted to 7 with a saturated aqueous solution of sodium bicarbonate, and the aqueous solution was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and then spin-dried to obtain 1.46 g of product, with a yield of 96%.

[0021] 1 H NMR (600MHz, DMSO-d6): δ9.79(s, 1H), 7.41(d, J=8.8Hz, 2H), 6.89(d, J=8.8Hz, 2H), 3.38(s, 3H)ppm.

[0022] Example 2: Preparation of p-nitrobenzyl chlorine formate (compound 2a) JPEG2025506061000006.jpg40148

[0023] p-Nitrobenzyl alcohol (0.168g, 1.1mmol) and BTC (0.296g, 1.0mmol) were dissolved in 20mL of anhydrous tetrahydrofuran, cooled in an ice-water bath, N,N-diisopropylethylamine (0.142g, 1.1mmol) was added, and the mixture was reacted for 30min under nitrogen gas protection. The mixture was then reacted at room temperature for 5h. After the reaction was completed, the reaction solution was concentrated, excess phosgene was removed with a small amount of anhydrous tetrahydrofuran, and anhydrous dichloromethane was added before being used directly in the next reaction.

[0024] Example 3: Preparation of 4-((methylnitroso)amino)phenyl(4-nitrobenzyl)carbonate (Compound I1) JPEG2025506061000007.jpg42142

[0025] Compound 1 (0.152 g, 1.0 mmol) and N,N-diisopropylethylamine (0.258 g, 2.0 mmol) were dissolved in 20 mL of anhydrous dichloromethane and cooled in an ice-water bath, and the anhydrous dichloromethane solution containing compound 2a obtained in Example 2 was slowly added dropwise to the above solution. After the completion of the addition, the ice-water bath was removed and the reaction was allowed to proceed at room temperature for 5 h. The reaction solution was washed three times each with 0.5 M dilute hydrochloric acid, water, and saturated saline, and the organic phase was separated, dried overnight over anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain a yellow solid, which was recrystallized with a mixed solvent of ethyl acetate and petroleum ether to obtain 285 mg of pale yellow crystals, with a yield of 86%.

[0026] 1H NMR (600MHz, DMSO-d6): δ8.28-8.30(m, 2H), 7.74-7.76(m, 2H), 7.70-7.72(m, 2H), 7.45-7.48(m, 2H), 5.46(s, 2H), 3.44(s, 3H)ppm.13C NMR (150MHz, DMSO-d6): δ153.15, 150.02, 147.87, 143.07, 140.37, 129.32, 124.15, 122.79, 121.20, 69.03, 32.14ppm.HRMS(m / z)(ESI):calcd for C15H14N3O6[M+H]+:332.08, found:332.11.

[0027] Example 4: Preparation of 4-((methylnitroso)amino)phenyl(2-chloro-4-nitrobenzyl)carbonate (Compound I2) JPEG2025506061000008.jpg41143

[0028] Referring to the method described in Examples 2 and 3, 2-chloro-4-nitrobenzyl alcohol was used as raw material to obtain compound I2, which was a pale yellow crystal, with a yield of 23%.

[0029] 1H NMR (600MHz, DMSO-d6): δ8.38-8.39(d, J=2.3Hz, 1H), 8.28-8.30(dd, 1H), 7.88-7.90(d , J=8.5Hz, 1H), 7.71-7.72(m, 2H), 7.46-7.48(m, 2H), 5.49(s, 2H), 3.44(s, 3H)ppm.13C NMR (150MHz, DMSO-d6): δ152.99, 149.95, 148.49, 140.41, 140.18, 133.62, 131 .17, 124.78, 122.96, 122.77, 121.25, 66.92, 32.20ppm.HRMS(m / z)(ESI):calcd for C15H13ClN3O6Na[M+Na]+:388.03, found:388.10.

[0030] Example 5: Preparation of 4-((methylnitroso)amino)phenyl(2-methyl-4-nitrobenzyl)carbonate (Compound I3) JPEG2025506061000009.jpg45162

[0031] Referring to the method described in Examples 2 and 3, 2-methyl-4-nitrobenzyl alcohol was used as raw material to obtain compound I3, which was a pale yellow crystal, with a yield of 45%.

[0032] 1H NMR (600MHz, DMSO-d6): δ8.15-8.16(d, J=2.2Hz, 1H), 8.10-8.14(dd, J=8.4, 2.4Hz, 1H), 7.71-7.73( d, J=2.2Hz, 1H), 7.68-7.70(m, 2H), 7.45-7.48(m, 2H), 5.44(s, 2H), 3.44(s, 3H), 2.47(s, 3H)ppm.13C NMR (150MHz, DMSO-d6): δ153.11, 150.01, 147.78, 141.17, 140.37, 139.16, 129.71 , 125.06, 122.80, 121.49, 121.21, 67.66, 32.17, 18.77ppm.HRMS(m / z)(ESI):calcd for C16H15N3O6[M+H]+:346.10, found:346.10.

[0033] Example 6: Preparation of 4-((methylnitroso)amino)phenyl(2-nitrobenzyl)carbonate (Compound I4) JPEG2025506061000010.jpg43136

[0034] Referring to the method described in Examples 2 and 3, 2-nitrobenzyl alcohol was used as raw material to obtain compound I4, which was a pale yellow crystal with a yield of 73%.

[0035] 1H NMR (600MHz, DMSO-d6): δ8.17-8.19(dd, 1H), 7.85-7.88(m, 1H), 7.79-7.80(m, 1H), 7.7 0-7.73(m, 2H), 7.68-7.69(m, 1H), 7.45-7.47(m, 2H), 5.65(s, 2H), 3.44(s, 3H)ppm.13C NMR (150MHz, DMSO-d6): δ153.04, 149.98, 147.82, 140.40, 134.85, 130.81, 130 .25, 130.06, 125.49, 122.77, 121.24, 67.09, 32.16ppm.HRMS(m / z)(ESI):calcd for C15H14N3O6[M+H]+:332.09, found:332.02..

[0036] Example 7: Preparation of 4-((methylnitroso)amino)phenyl(2-nitro-4-chlorobenzyl)carbonate (Compound I5) JPEG2025506061000011.jpg43146

[0037] Referring to the method described in Examples 2 and 3, 2-nitro-4-chlorobenzyl alcohol was used as raw material to obtain compound I5, which was a pale yellow crystal, with a yield of 46%.

[0038] 1H NMR (600MHz, DMSO-d6): δ8.26-8.27(d, J=2.2Hz, 1H), 7.94-7.96(dd, 1H), 7.81-7.83(d , J=8.4Hz, 1H), 7.70-7.72(m, 2H), 7.45-7.46(m, 2H), 5.62(s, 2H), 3.44(s, 3H)ppm.13C NMR (150MHz, DMSO-d6): δ152.94, 149.96, 148.47, 140.42, 134.51, 134.21, 131 .84, 129.81, 125.32, 122.75, 121.24, 66.53, 32.15ppm.HRMS(m / z)(ESI):calcd for C15H13ClN3O6Na[M+Na]+:388.03, found:388.00.

[0039] Example 8: Preparation of 4-((methylnitroso)amino)phenyl(2-nitro-4-bromobenzoyl)carbonate (Compound I6) JPEG2025506061000012.jpg44135

[0040] Referring to the method described in Examples 2 and 3, 2-nitro-4-bromobenzyl alcohol was used as raw material to obtain compound I6, which was a pale yellow crystal, with a yield of 33%.

[0041] 1H NMR (600MHz, DMSO-d6): δ8.36-8.37(d, J=2.1Hz, 1H), 8.07-8.09(dd, 1H), 7.73-7.75(d , J=8.3Hz, 1H), 7.70-7.72(m, 2H), 7.44-7.46(m, 2H), 5.60(s, 2H), 3.44(s, 3H)ppm.13C NMR (150MHz, DMSO-d6): δ152.94, 149.96, 148.49, 140.42, 137.45, 131.94, 13 0.19, 128.03, 122.75, 122.16, 121.24, 66.58, 32.15.HRMS(m / z)(ESI):calcd for C15H13BrN3O6[M+H]+:431.98, found:431.90.

[0042] Example 9: Preparation of 4-((methylnitroso)amino)phenyl(2-nitro-4-methylbenzyl)carbonate (Compound I7) JPEG2025506061000013.jpg38129Referring to the methods described in Examples 2 and 3, 2-nitro-4-methylbenzyl alcohol is used as raw material to obtain compound I7, which is pale yellow crystal, with a yield of 51%.

[0043] 1H NMR (600MHz, DMSO-d6): δ8.00-8.01(m, 1H), 7.70-7.72(m, 2H), 7.66-7.67(d, J =0.9Hz, 2H), 7.43-7.46(m, 2H), 5.60(s, 2H), 3.44(s, 3H), 2.44(s, 3H)ppm.13C NMR (150MHz, DMSO-d6): δ153.04, 149.99, 147.77, 140.57, 140.38, 135.29, 130.22 , 127.75, 125.56, 122.74, 121.20, 67.02, 32.12, 20.74ppm.HRMS(m / z)(ESI):calcd for C16H16N3O6[M+H]+:346.10, found:345.90.

[0044] Example 10: Preparation of 4-((methylnitroso)amino)phenyl(2-nitro-5-chlorobenzyl)carbonate (Compound I8) JPEG2025506061000014.jpg48129

[0045] Referring to the method described in Examples 2 and 3, 2-nitro-5-chlorobenzyl alcohol was used as raw material to obtain compound I8, which was a pale yellow crystal, with a yield of 37%.

[0046] 1H NMR (600MHz, DMSO-d6): δ8.21-8.23(d, J=8.8Hz, 1H), 7.83-7.84(d, J=2.3Hz, 1H), 7.76 -7.79(dd, 1H), 7.71-7.73(m, 2H), 7.46-7.48(m, 2H), 5.65(s, 2H), 3.44(s, 3H)ppm.13C NMR (150MHz, DMSO-d6): δ152.82, 149.98, 146.36, 140.43, 139.48, 133.27, 130 .05, 129.67, 127.63, 122.76, 121.23, 66.56, 32.14ppm.HRMS(m / z)(ESI):calcd for C15H13ClN3O6Na[M+Na]+:388.03, found:388.00.

[0047] Example 11: Preparation of 4-((methylnitroso)amino)phenyl(2-nitro-5-methylbenzyl)carbonate (Compound I9) JPEG2025506061000015.jpg49128

[0048] Referring to the method described in Examples 2 and 3, 2-nitro-5-methylbenzyl alcohol was used as raw material to obtain compound I9, which was a pale yellow crystal, with a yield of 48%.

[0049] 1H NMR (600MHz, DMSO-d6): δ8.09-8.11(d, J=8.4Hz, 1H), 7.72-7.73(d, J=2.2Hz, 1H), 7.71-7.72(d, J=2.3Hz, 1H), 7.57-7.58(m, 1 H), 7.48-7.49(m, 1H), 7.46-7.47(d, J=2.3Hz, 1H), 7.45-7.46(d, J=2.2Hz, 1H), 5.63(s, 2H), 3.44(s, 3H), 2.47(s, 3H)ppm.13C NMR (150MHz, DMSO-d6): δ153.00, 150.01, 145.93, 145.49, 140.40, 130.95, 130.47 , 130.26, 125.70, 122.78, 121.24, 67.21, 32.15, 21.53ppm.HRMS(m / z)(ESI):calcd for C16H15N3O6Na[M+Na]+:368.09, found:368.10.

[0050] Example 12: Detection of NO release levels after compounds act on H9c2 cardiomyocytes in normoxia or hypoxia by the Griess method Cardiomyocytes H9c2 were resuscitated and cultured in DMEM medium, then placed in a carbon dioxide incubator to allow them to adhere to the wall and grow. After the cells reached an appropriate density, they were attached to a porous plate, placed in a carbon dioxide incubator, and cultured for 24 h under conditions of 37°C, 5% CO2 and saturated humidity until the cells adhered to the wall. The supernatant was discarded, 2 mL of fresh DMEM medium was added, and a certain concentration (5, 10, 15 μM) of the compound and the control drug isosorbide dinitrate were added. After 4 h of incubation in a normoxic incubator (37°C, 21% O2) or hypoxic incubator (37°C, 1% O2), the supernatant was removed, and the cells in the plate were collected in 1.5 mL centrifuge tubes, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 1 mL of PBS was added to resuspend the cells, centrifuged again, the PBS was discarded, 100 μL of cell lysis solution (for NO measurement) was added to the centrifuge tube, left on ice for half an hour, and centrifuged at 13300 rpm at 4°C for 5 min to collect the supernatant. 50 μL of the supernatant was taken into a 96-well plate, and three parallel groups were set up for each sample, and 50 μL of Griess reagent I and 50 μL of Griess reagent II were added sequentially. The OD value of each well was measured using a full-wavelength microplate reader at a wavelength of 540 nm, and the NO concentration was calculated from the standard curve. The results are shown in Tables 1 and 2.

[0051] Table 1 NO release in H9c2 cardiomyocytes under normoxia 4 hours after treatment with compounds JPEG2025506061000016.jpg100170*.NO release = measured NO concentration / calculated NO concentration. Data are expressed as the mean SD of three independent experiments: *p<0.05, **p<0.01, a. Represents unmeasured, b. Calculated as 1 / 2 of the amount released.

[0052] Table 2 NO release 4 hours after treatment of H9c2 cardiomyocytes with compounds under hypoxic conditions JPEG2025506061000017.jpg99170*.NO release = measured NO concentration / calculated NO concentration. Data are expressed as the mean SD of three independent experiments: *p<0.05, **p<0.01, a. Represents unmeasured, b. Calculated as 1 / 2 of the amount released.

[0053] According to the data in Tables 1 and 2, the amount of NO released in cells after treatment of cardiomyocytes with different concentrations of compounds under normoxia was very small, all of which were lower than the maximum release of compound I2 (<3.34%, 10 μM); however, under hypoxia, compounds I1-I4 at different concentrations, in addition to compounds I5-I9, could effectively induce the release of NO in cells, with the release amount ranging from 20.15% to 45.45%, and appeared as hypoxia-activated NO donors. The control product, isosorbide dinitrate (ISDN), released a certain amount of NO after treatment of cardiomyocytes, regardless of whether it was under normoxia or hypoxia, and the amount of NO released in cells under normoxia was relatively large, reaching 41.63% (10 μM), and could induce the release of NO in cells even under hypoxia, but the release amount was smaller than that under normoxia and lower than the release level of compounds I2 and I3.

[0054] Example 13: Improving effect of compounds on survival rate of cardiomyocytes H9c2 under normoxia or hypoxia The cells were washed twice with 5 mL of PBS, 1 mL of tryptin was added to digest the monolayer cultured cardiomyocytes H9c2, and a single cell suspension was prepared in a culture medium containing serum. 5,000 to 10,000 cells per well were inoculated into a 96-well culture plate, with 100 μL of medium volume per well, and the edge well was filled with sterilized pure water. The culture plate was transferred to a carbon dioxide incubator and cultured for 24 h under conditions of 37 °C, 5% CO2 and saturated humidity until the cells attached to the wall, 100 μL of medium in the well was aspirated, 50 μL of fresh medium was added, and different concentrations of the compound and the control drug isosorbide dinitrate were given. 50 μL of the drug was added per well, and three parallel groups and one normoxic non-administration group were set up as controls. (The concentration gradient was 10, 1, 0.1, 0.01, and 0.001 μM.) The normoxic culture group was incubated in a normal incubator at 5% CO2 and 37℃ for 4h, while the hypoxic group was incubated in a hypoxic incubator (O2 concentration ≦1%) at 37℃ for 4h. After incubation was completed, 10μL of CCK8 reagent was added per well and incubation was continued in a normoxic incubator for 4h. After incubation was completed, the absorbance value of each well was measured at 450nm using a microplate reader, and the cell viability of cardiomyocytes at each concentration was calculated. The results are shown in Figure 1a and Figure 1b.

[0055] Under normoxic conditions (see Figure 1a), compounds I1-I9 showed no improvement on cell viability with increasing concentration, except compound I4 showed slight improvement on cell viability at 1μM, and on the contrary, they also affected cell viability at high concentration of 10μM and showed relatively small toxicity. Under hypoxic conditions (see Figure 1b), compounds I1-I4 all significantly restored hypoxic cell viability within the concentration range of 0.001-10μM and improved with increasing compound concentration, while compounds I5-I9 had no obvious effect on restoring hypoxic cell viability. The control drug isosorbide dinitrate showed no improvement on cell viability under normoxic conditions, and showed a certain ability only at high concentration under hypoxic conditions, but was inferior to compounds I1-I4. Combining the data in Tables 1 and 2, these hypoxia-activated NO donor compounds I1-I4 can effectively release NO under hypoxic conditions and improve the survival rate of cardiomyocytes at a certain concentration range, and their activity is superior to that of the conventional drug isosorbide dinitrate. This beneficial effect was also verified in animal experiments.

[0056] Example 14: Detection of NO release level in hypoxic model mouse heart of compound I1 by NO detection kit (a) Drugs and reagents and laboratory animals NO detection kit (Biyuntian Biotechnology Co., Ltd., Shanghai), hypoxia modeling and administration mice, blank control group mouse heart tissue homogenate, 6-8 week-old male Kunming mice.

[0057] (b) Experimental principle NO is chemically active and is converted to NO3 in the body. - and NO2 - is rapidly converted to NO2 - is further NO3 - It is converted to NO3 using Griess reagent. - NO2 - Reduced to NO2 - has a characteristic absorption at 540 nm, and the absorbance at 540 nm was detected using a microplate reader, and the NO concentration was calculated using a standard curve.

[0058] (c) Experimental procedure (1) Hypoxia model (85 mg / kg isoproterenol), the treatment group was intraperitoneally injected with 20 mg / kg compound I1 before hypoxia modeling; (2) Centrifuge at 12,000 rpm for 15 min to collect cardiac tissue homogenate proteins. (3) The working solution for the NO detection kit was prepared, and the working solution was added to the proteins of each group. After incubation, the NO content of each group was analyzed by detecting it with a microplate reader. The results are shown in Figure 2.

[0059] As shown in FIG. 2, after hypoxia modeling and administration to mice, the intracardiac NO level in the compound I1 group was significantly higher than that in the other two groups.

[0060] Example 15: Study of the regulatory effect of compound I1 on the expression level of myocardial injury marker protein in myocardial hypoxic mice by Western blot analysis (a) Drugs and reagents and laboratory animals Western Blot gel preparation kit (Biyuntian Biotechnology Co., Ltd., Shanghai), BCA detection kit for protein quantification (Biyuntian Biotechnology Co., Ltd., Shanghai), ionization buffer, wet transfer liquid, primary antibody (anti-TSC2-P, Abgent, 1:1000, A-AP3825a, anti-mTORC1, Abcam, 1:1000, ab120224, anti-GAPDH, Bioworld Technology, 1:500, AP0063), secondary antibody (Goat Anti-Rabbit IgG(H+L)HRP, 1:3000, Bioworld Technology, BS13278), 6-8 week old male Kunming mice.

[0061] (b) Experimental principle The changes in protein expression levels were analyzed based on Western Blot bands.

[0062] (c) Experimental procedure (1) Mouse heart tissue protein was extracted, centrifuged at 12,000 rpm for 15 min to collect the protein, quantified using a BCA kit, added to the loading buffer in boiling water, boiled for 5 min, and reserved. (2) Prepare an SDS-PAGE gel, load the samples and separate the protein bands; (3) After transferring the protein bands to a PVDF membrane, the membrane was blocked with milk, incubated with primary and secondary antibodies, and visualized using a chemiluminescent developer to analyze the changes in protein expression in each group. The results are shown in Figure 3.

[0063] As shown in Figure 3, protein samples were extracted from the hearts of blank mice, hypoxia model mice, and administration + hypoxia modeling mice, and Western blot experiments were performed. Compared with the blank group, the decline in TSC2-P expression in hypoxia model mice caused a significant increase in mTORC1 protein expression, indicating that the model group had obvious myocardial damage. The mTORC1 expression in the hearts of mice pretreated with compound I at 120 mg / kg was restored to a level close to that of the normal group, indicating that this compound has an excellent anti-myocardial hypoxic injury effect.

Claims

1. An NO donor compound having the structure of Formula I: Where: R １ is a nitro group, R ２ is hydrogen, halogen or C １ ~C ４ is an alkyl group, R ３ is hydrogen, halogen or C １ ~C ４ is an alkyl group, R ２ is a nitro group, R １ is hydrogen, halogen or C １ ~C ４ is an alkyl group, R ３ is hydrogen, halogen or C １ ~C ４ An NO donor compound, which is an alkyl group.

2. In the above structure, R １ is a nitro group, R ２ is hydrogen, halogen or a methyl group; R ３ The NO donor compound of claim 1, wherein is hydrogen.

3. The NO donor compound described in claim 1, characterized in that it is any one compound selected from the following:

4. A method for producing the NO donor compound according to any one of claims 1 to 3, comprising the steps of: subjecting compound 1 to compound 2 to an acylation reaction to obtain compound I; Here, R １ , R ２ , R ３ is defined as in any one of claims 1 to 3.

5. A pharmaceutical composition comprising an NO donor compound according to any one of claims 1 to 3 and a pharma- ceutically acceptable carrier.

6. 4. Use of the NO donor compound according to any one of claims 1 to 3 in the manufacture of a drug for treating myocardial hypoxic injury disease.

7. Use of the pharmaceutical composition according to claim 5 in the manufacture of a drug for treating myocardial hypoxic injury disease.

8. The use according to claim 6 or 7, characterized in that the compound is a hypoxia-activated NO donor compound.

9. 8. The use according to claim 6 or 7, characterized in that the drug is used to improve the survival rate of cardiomyocytes under hypoxic conditions.

10. 8. The use according to claim 6 or 7, characterized in that the disease is coronary heart disease.

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